Natural nano-structural attapulgite clay was purified by mechanical stirring with the aid of ultrasonic wave and its structure and morphology was investigated by XRD and transmission electron microscopy (TEM). Cytochrome c was immobilized on attapulgite modified glassy carbon electrode. The interaction between Cytochrome c and attapulgite clay was examined by using UV-vis spectroscopy and electrochemical methods. The direct electron transfer of the immobilized Cytochrome c exhibited a pair of redox peaks with formal potential (E-0') of about 17 mV (versus SCE) in 0.1 mol/L, pH 7.0, PBS. The electrode reaction showed a surface-controlled process with the apparent heterogeneous electron transfer rate constant (k(s)) of 7.05 s(-1) and charge-transfer coefficient (alpha) of 0.49. Cytochrome c immobilized on the attapulgite modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide (H2O2). The calculated apparent Michaelis-Menten constant (K-m(app)) was 470 mu mol/L, indicating a high catalytic activity of Cytochrome c immobilized on attapulgite modified electrode to the reduction of H2O2. Based on these, a third generation of reagentless biosensor can be constructed for the determination of H2O2. (c) 2006 Elsevier Ltd. All rights reserved.